In Phase I we propose to create a library of tagged knock-out mutations in the Saccharopolyspora erythraea genome, and to find at least one new mutation that affects erythromycin biosynthesis. In Phase II we plan a comprehensive screen of the library to find all mutations that affect erythromycin production. In Phase III metabolic engineering strategies will be developed to create improved strains. Although the 60 kb erythromycin biosynthetic gene cluster has already been cloned and characterized, no regulatory genes have been found within it. Since this cluster makes up less than 1% of the genome, we propose a comprehensive search of the remaining portion of the genome for regulatory mutations. We hypothesize based on results in closely related organisms, that many genes affecting production will be found. Traditional mutate-and-screen methods have been successful in producing these mutations in Sac. erythraea, but not a single strain improvement gene has yet been identified using this approach. In contrast, the proposed study should lead to the identification of the complete set of genes affecting erythromycin production. Rational metabolic engineering of these genes has the potential of generating strains that are much better than those currently available. PROPOSED COMMERCIAL APPLICATIONS: Commercial strains for the production of the bulk pharmaceutical erythromycin are responsible for the production of a compound with a market value of more than $600 million per year, world-wide. Any strain that is significantly superior to existing strains would therefore be of significant economic value. The development of new first and second generation erythromycin derivatives means the market for erythromycin as a chemical intermediate will continue to grow well into the next century.